Predicted Environmental Risk Assessment of Antimicrobials with Increased Consumption in Portugal during the COVID-19 Pandemic; The Groundwork for the Forthcoming Water Quality Survey.

The environmental release of antimicrobial pharmaceuticals is an imminent threat due to ecological impacts and microbial resistance phenomena. The recent COVID-19 outbreak will likely lead to greater loads of antimicrobials in the environment. Thus, identifying the most used antimicrobials likely to pose environmental risks would be valuable. For that, the ambulatory and hospital consumption patterns of antimicrobials in Portugal during the COVID-19 pandemic (2020-2021) were compared with those of 2019. A predicted risk assessment screening approach based on exposure and hazard in the surface water was conducted, combining consumption, excretion rates, and ecotoxicological/microbiological endpoints in five different regions of Portugal. Among the 22 selected substances, only rifaximin and atovaquone demonstrated predicted potential ecotoxicological risks for aquatic organisms. Flucloxacillin, piperacillin, tazobactam, meropenem, ceftriaxone, fosfomycin, and metronidazole showed the most significant potential for antibiotic resistance in all analysed regions. Regarding the current screening approach and the lack of environmental data, it is advisable to consider rifaximin and atovaquone in subsequent water quality surveys. These results might support the forthcoming monitorisation of surface water quality in a post-pandemic survey.

Parasiticides: Weapons for Controlling Microbial Vector-Borne Diseases in Veterinary Medicine; The Potential of Ethnobotanic/Phytoparasiticides: An Asset to One Health.

Some ectoparasites are vectors of illness-causing bacteria and viruses, and these are treated with antibiotic and antiviral drugs, which eventually contribute to the excessive use of antimicrobials. Therefore, the control of ectoparasites is crucial, and the challenge will be to manage them in a sustainable way. Data from a preliminary ethnobotanical survey was reanalyzed to obtain information on the use of various plant species in companion animals and livestock as ectoparasiticides. The survey responses were reviewed for traditional use of plants as ectoparasiticides, and cross-sectional bibliographic research was undertaken. The following plants were selected among the nine mentioned plants: Juglans regia, Daphne gnidium and Ruta graveolens, which have the most potential to be developed as veterinary ectoparasiticides. Moreover, the absence of published data for Plantago lanceolata and Cistus populifolius suggests that their traditional use as ectoparasiticides is noted here for the first time. In summary, these plants could give promising plant-derived veterinary ectoparasiticides that, ultimately, will help reduce and even avoid the excessive use of antimicrobials.

Identification of Antibiotics in Surface-Groundwater. A Tool towards the Ecopharmacovigilance Approach: A Portuguese Case-Study.

Environmental monitoring, particularly of water, is crucial to screen and preselect potential hazardous substances for policy guidance and risk minimisation strategies. In Portugal, extensive data are missing. This work aimed to perform a qualitative survey of antibiotics in surface- groundwater, reflecting demographic, spatial, consumption and drug profiles during an observational period of three years. A passive sampling technique (POCIS) and high-resolution chromatographic system were used to monitor and analyse the antibiotics. The most frequently detected antibiotics were enrofloxacin/ciprofloxacin and tetracycline in surface-groundwater, while clarithromycin/erythromycin and sulfamethoxazole were identified only in surface water. The detection of enzyme inhibitors (e.g., tazobactam/cilastatin) used exclusively in hospitals and abacavir, a specific human medicine was also noteworthy. North (Guimarães, Santo Tirso and Porto) and South (Faro, Olhão and Portimão) Portugal were the regions with the most significant frequency of substances in surface water. The relatively higher detection downstream of the effluent discharge points compared with a low detection upstream could be attributed to a low efficiency in urban wastewater treatment plants and an increased agricultural pressure. This screening approach is essential to identify substances in order to perform future quantitative risk assessment and establishing water quality standards. The greatest challenge of this survey data is to promote an ecopharmacovigilance framework, implement measures to avoid misuse/overuse of antibiotics and slow down emission and antibiotic resistance.

Human pharmaceuticals in Portuguese rivers: The impact of water scarcity in the environmental risk.

Pharmaceuticals occurrence and environmental risk assessment were assessed in Portuguese surface waters, evaluating the impact of wastewater treatment plants (WWTPs) and river flow rates. Twenty three pharmaceuticals from 6 therapeutic groups, including metabolites and 1 transformation product, were analysed in 72 samples collected from 20 different sites, upstream and downstream the selected WWTPs, in two different seasons. Analysis was performed by solid phase extraction followed by liquid chromatography coupled to tandem mass spectroscopy. Pharmaceuticals were detected in 27.8% of the samples. Selective serotonin reuptake inhibitors (SSRIs), anti-inflammatories and antibiotics presented the highest detection frequencies (27.8, 23.6 and 23.6%, respectively) and average concentrations (37.9, 36.1 and 33.5ngL-1, respectively). When assessing the impact of WWTPs, an increase of 21.4% in the average concentrations was observed in the samples located downstream these facilities, when compared with the upstream samples. Increased detection frequencies and concentrations were observed at lower flow rates, both when comparing summer and winter campaigns and by evaluating the different rivers. Risk quotients (RQs) higher than one were found for two pharmaceuticals, concerning two trophic levels. However, since Iberian rivers are highly influenced by water scarcity, in drought periods, the flow rates in these rivers can decrease at least ten times from the lowest value observed in the sampling campaigns. In these conditions, RQs higher than 1 would be observed for 5 pharmaceuticals, additionally, all the detected pharmaceuticals (11) would present RQs higher than 0.1. These results emphasize that the river flow rate represents an important parameter influencing pharmaceuticals concentrations, highlighting the ecotoxicological pressure, especially due to water scarcity in drought periods. This should be a priority issue in the environmental policies for minimizing its impact in the aquatic environment.

SSRIs antidepressants in marine mussels from Atlantic coastal areas and human risk assessment.

Selective serotonin reuptake inhibitors (SSRIs) are among the most prescribed antidepressants nation and worldwide. Their presence in the aquatic environment is known, causing growing pressures in heavily populated coastal areas. For instance, disruption of invertebrate endocrine systems has been described. Since discrete seawater sampling may not be suitable to evaluate such environmental risk, we aimed to assess SSRIs and metabolites in wild Mytilus galloprovincialis, and assess risk to human health. A total of 1225 mussels were sampled during one year along the Portuguese Atlantic coast. Each freeze-dried pool of 25 specimens was analyzed, in duplicate, by solid phase extraction (SPE) and liquid chromatography with tandem mass spectrometry (LC-MSn). Approximately 70% of the samples were contaminated with at least one, and up to 4 from the 7 targeted compounds. The cumulative SSRIs and metabolites content in mussels ranged between 3.17 and 33.93ngg-1 dry weight, in a mean level of 14.68±9.69ngg-1. Norfluoxetine was the most recurring SSRI and the one with the highest mean concentration, 13.52ngg-1. Although some differences were found between different sampling periods and locations, those were not statistically significant (p>0.05) and no correlation was found between wet weight and mean contamination levels. Regarding risk to human health following consumption of these filter-feeders, when considering the average sample contamination of positive samples and the average Portuguese and European consumption, the ratio between the estimated daily intake (EDI) and the acceptable daily intake (ADI) ranged from 0.45 to 5.50%, and from 1.60 to 19.41%, respectively. Therefore, although the resultant risk is lower for the Portuguese consumer than for the average EU consumer, apparently, no appreciable risk to human health is expected.

A critical evaluation of different parameters for estimating pharmaceutical exposure seeking an improved environmental risk assessment.

A critical evaluation of the European Medicines Agency (EMA) Guideline on Environmental Risk Assessment (ERA) was performed on 16 of Portugal's most consumed pharmaceuticals in wastewater effluents (WWEs), the main route for aquatic contamination. The predicted environmental concentrations (PECs) were formulated based on the Guideline, after incorporating several refinements. The best approach was selected by comparing the measured environmental concentrations (MECs) to the PECs in WWEs. Finally, risk was assessed by comparing PECs to predicted no-effect concentrations (PNECs). The results showed that the default value of the penetration factor (Fpen) used by the EMA (0.01) was surpassed and that national consumption and excretion data were the two most important parameters for PEC calculations. The risk quotient between PECs and PNECs was higher than 1 for 12 pharmaceuticals, indicating a risk to all three trophic levels of aquatic organisms (algae, daphnids and fish). To improve the current ERA framework, suggestions were made for incorporating consumption and excretion data, changing the default value of Fpen to 0.04 and adding a safety factor of 10. Moreover, this evaluation should be performed for pharmaceuticals already on the market, and future ERAs should incorporate a risk-benefit analysis, an important risk-management step.

Uptake, accumulation and metabolization of the antidepressant fluoxetine by Mytilus galloprovincialis.

Fluoxetine, a selective serotonin re-uptake inhibitor (SSRI) antidepressant, is among the most prescribed pharmaceutical active substances worldwide. This study aimed to assess its accumulation and metabolization in the mussel Mytillus galloprovincialis, considered an excellent sentinel species for traditional and emerging pollutants. Mussels were collected from Ria Formosa Lagoon, Portugal, and exposed to a nominal concentration of fluoxetine (75 ng L(-1)) for 15 days. Approximately 1 g of whole mussel soft tissues was extracted with acetonitrile:formic acid, loaded into an Oasis MCX cartridge, and fluoxetine analysed by liquid chromatography with tandem mass spectrometry (LC-MSn). After 3 days of exposure, fluoxetine was accumulated in 70% of the samples, with a mean of 2.53 ng g(-1) dry weight (d.w.) and norfluoxetine was only detected in one sample (10%), at 3.06 ng g(-1) d.w. After 7 days of exposure, the accumulation of fluoxetine and norfluoxetine increased up to 80 and 50% respectively, and their mean accumulated levels in mussel tissues were up to 4.43 and 2.85 ng g(-1) d.w., respectively. By the end of the exposure period (15 days), both compounds were detected in 100% of the samples (mean of 9.31 and 11.65 ng g(-1) d.w., respectively). Statistical analysis revealed significant accumulation differences between the 3rd and 15th day of exposure for fluoxetine, and between the 3rd and 7th against the 15th day of exposure for norfluoxetine. These results suggest that the fluoxetine accumulated in mussel tissues is likely to be metabolised into norfluoxetine with the increase of the time of exposure, giving evidence that at these realistic environmental concentrations, toxic effects of fluoxetine in mussel tissues may occur.

Assessing environmental risk of pharmaceuticals in Portugal: An approach for the selection of the Portuguese monitoring stations in line with Directive 2013/39/EU.

In line with the Directive 2013/39/EU the most representative surface waters, regarding pharmaceuticals contamination, were selected based on a Portuguese nationwide monitoring exercise. To meet this purpose, and given that wastewater treatment plants (WWTPs) are regarded as the major point sources of pharmaceuticals environmental contamination, the occurrence, fate and environmental risk assessment (ERA) of eleven of the most consumed pharmaceuticals, belonging to several therapeutic classes were assessed in 15 WWTPs (influents (WWIs) and effluents (WWEs)), from five different regions during one year (4 sampling campaigns). Results showed that all samples were contaminated with at least 1, and up to 8 from the 11 targeted pharmaceuticals. The highest concentrations observed were 150 and 33 µg L(-1) for WWI and WWE, respectively. Regarding temporal and spacial influence, winter, Alentejo, Algarve and Center regions presented higher mass loads. The ERA posed by 7 of the selected pharmaceuticals presented a risk quotient higher than 1 to the three trophic levels. Our findings highlighted that the rivers Mondego, Tagus, Ave, Trancão, Fervença and Xarrama should be selected as surface water monitoring stations. This study gives a good overview on pharmaceuticals contamination in WWTPs and its impact on surface waters in Portugal. Thus, a more integrative approach to rank and prioritize pharmaceuticals, based on an integrated assessment of ERA and exposure of surface water, was provided to support the future selection of the 6 most representative monitoring stations in Portugal, as required by the above mentioned directive.

Fluoroquinolones and Tetracycline Antibiotics in a Portuguese Aquaculture System and Aquatic Surroundings: Occurrence and Environmental Impact.

The growth of aquaculture over the past few years is widely recognized as one of the main sources of antibiotics, mainly fluoroquinolones (FQ) and tetracyclines (TC), in the aquatic environment, consequently, increasing the risk of the emergence of antibiotic bacterial resistance and promoting the spread of resistant genes. This study aimed to (1) develop and validate a multiresidue method for determination and quantification of ciprofloxacin (CIP), difloxacin (DIFL), enrofloxacin (ENR), norfloxacin (NOR), sarafloxacin (SARA), and oxytetracycline (OXY) in aquaculture waters and surrounding water bodies and (2) provide the first Portuguese data to utilize in assessment of risk of adverse effects. In addition, the potential environmental impact posed by these antibiotics to aquatic organisms, belonging to different trophic levels, when exposed to the studied aquaculture waters was also assessed. The analytical strategy comprised of solid-phase extraction (SPE) through Oasis HLB cartridges, and detection and quantification by liquid chromatography with tandem mass spectrometry (LC/MS(n)). Method detection limits (MDL) and method quantification limits (MQL) were in the range of 0.7-3 ng/L and 2.4-10 ng/L, respectively. Recoveries varied between 57.4 and 122.8%. The method was applied to 31 water samples collected from an aquaculture and surrounding water bodies located in north of Portugal. Residues of all antibiotics, except SARA and DIFL, were detected at concentrations ranging from 3 to 75.1 ng/L. Norfloxacin was the antibiotic present at highest frequency and concentration. Regarding the environmental impact assessment (EIA), a risk quotient higher than 1 was observed for NOR.

Environmental impact of pharmaceuticals from Portuguese wastewaters: geographical and seasonal occurrence, removal and risk assessment.

The occurrence, fate, geographical and seasonal influence and environmental risk assessment of eleven of the most consumed pharmaceuticals in Portugal were studied in wastewater treatment plants (WWTPs) influents and (WWI) and effluents (WWE). WWI and WWE samples, from two sampling campaigns (spring and summer), in 2013, were evaluated in 15 different WWTPs across the country, by solid phase extraction (SPE) and liquid chromatography coupled with tandem mass detection (LC-MS-MS). Lipid regulators were the most frequently found in WWI and WWE (184.1 and 22.3mg/day/1000 inhab., respectively), followed by anti-inflammatories (1339.4 and 15.0mg/day/1000 inhab., respectively), and antibiotics (330.7 and 68.6 mg/day/1000 inhab., respectively). Anxiolytics were the least detected with 3.3 and 3.4 mg/day/1000 inhab. in WWI and WWE, respectively. The mass loads, both in WWI and WWE, were higher in summer than those found during the spring season, being remarkable the high values registered in a region where population triplicates in this time of the year. The mean removal efficiency achieved was of 94.5%, nonetheless, between the different therapeutic groups, as well as within each group, important variations in removal were observed, going from not eliminated to 100%. In the summer higher efficiencies were observed regarding lipid regulators and antibiotics. Furthermore, an important outcome was the evaluation, by means of risk quotients (RQs), of the potential ecotoxicological risk posed by the selected pharmaceuticals to different aquatic organisms, exposed to the effluents studied. Ciprofloxacin, bezafibrate, gemfibrozil, simvastatin and diclofenac showed RQs higher than one, being expected that these pharmaceuticals might pose a threat to the three trophic levels (algae, daphnids and fish) evaluated. These results highlight the importance of these monitoring studies, as required by the Directive 2013/39/EU, in order to minimize their aquatic environmental contamination and support future prioritization measures.

Reviewing the serotonin reuptake inhibitors (SSRIs) footprint in the aquatic biota: uptake, bioaccumulation and ecotoxicology.

Selective serotonin re-uptake inhibitors (SSRIs) antidepressants are amongst the most prescribed pharmaceutical active substances throughout the world. Their presence, already described in different environmental compartments such as wastewaters, surface, ground and drinking waters, and sediments, and their remarkable effects on non-target organisms justify the growing concern about these emerging environmental pollutants. A comprehensive review of the literature data with focus on their footprint in the aquatic biota, namely their uptake, bioaccumulation and both acute and chronic ecotoxicology is presented. Long-term multigenerational exposure studies, at environmental relevant concentrations and in mixtures of related compounds, such as oestrogenic endocrine disruptors, continue to be sparse and are imperative to better know their environmental impact.

Profiling serotonin reuptake inhibitors (SSRIs) in the environment: trends in analytical methodologies.

Selective serotonin reuptake inhibitor (SSRI) antidepressants are among the most prescribed pharmaceuticals worldwide and are ubiquitous in different areas of the environment. There are few reliable and specific analytical methods for the detection and quantification of SSRIs in environmental samples. The majority of the reported methods generally involve cleanup procedures that usually employ solid-phase extraction (SPE) followed by liquid chromatography with tandem mass spectrometry detection (LC-MS-MS). Herewith, a comprehensive overview of the described analytical methods available for the determination of SSRIs in environmental samples is provided. We address all steps involved in the analytical procedures, with the inherent advantages and disadvantages of each discussed.

A one-year follow-up analysis of antidepressants in Portuguese wastewaters: occurrence and fate, seasonal influence, and risk assessment.

The occurrence, fate, seasonal influence and environmental risk assessment of four selective serotonin re-uptake inhibitors (SSRIs) antidepressants, citalopram, fluoxetine, paroxetine and sertraline, were studied in 15 different wastewater treatment plants (WWTPs) across Portugal. Influent and effluent samples from four sampling campaigns, in 2013, were extracted through Oasis HLB cartridges, and quantified through liquid chromatography with tandem mass spectrometry (LC-MSn).Results showed that citalopram was the SSRI most frequently found, both in influents and in effluents, with mean mass loads ranging between 14.56 and 9.51 mg/day/1000 inhabitants, respectively. Fluoxetine and sertraline were only detected in influent samples, in lower mean mass loads (14.60 and 1.36 mg/day/1000 inhab., respectively), whereas paroxetine was found in influent and effluent samples (12.61 and 18.90 mg/day/1000 inhab., respectively). WWTPs were not capable of completely removing these pharmaceuticals; nonetheless, the mean removal efficiency was 82.24%. Removal efficiency was lower in winter (74.21%), summer (72.02%), and autumn (81.19%), when compared to spring (100%). Our results translate the variations in SSRI prescription and use between the five Portuguese regions in study. Influent contaminated samples were found in WWTPs from Lisbon, Alentejo, Center and North (28.25, 19.01, 16.55 and 6.98 mg/day/1000 inhab., respectively). In the Algarve region no contaminated samples were found. A seasonal pattern in the presence of SSRIs in influent wastewaters was observed. The SSRIs mass loads in influent wastewaters were higher in autumn, followed by spring, winter, and summer. Finally, the potential ecotoxicological risk posed by SSRIs to different trophic levels of aquatic organisms, exposed to the effluent wastewaters studied was evaluated by means of risk quotients (RQ). Citalopram and paroxetine, the only SSRIs found in these samples, presented RQ lower than 1. According to the results, algae appeared to be the most sensitive followed by fish and daphnids.

Selective serotonin re-uptake inhibitors (SSRIs) in the aquatic environment: an ecopharmacovigilance approach.

Selective serotonin re-uptake inhibitors (SSRIs) antidepressants are among the most prescribed pharmaceutical active substances throughout the world. The occurrence of these widely used compounds in different environmental compartments (wastewaters, surface, ground and drinking waters, and sediments), justify the growing concern about these emerging environmental pollutants. Viewing an ecopharmacovigilance approach, a comprehensive discussion of the state of the art regarding different contamination sources, fate, degradation and occurrence is presented. Information on the current distribution levels and fate in different environmental matrices continues to be sparse and measures are imperative to improve awareness and encourage precautionary actions to minimize SSRIs' environmental impact.